The goals of the proposed research are to gain a better understanding of the factors involved in transcription by RNA Polymerase II (RNApo II), and to elucidate the mechanisms underlying this process. Transcription by RNA pol II is an essential step in expression of protein coding genes. In vitro, accurate initiation of transcription can be reconstituted with the general transcription factors and RNA pol II. However, in vivo the process is clearly more complex as the transcription machinery must interact with regulatory faces as well as contend with the chromatin template. To obtain a more complete understanding of the transcription machinery, I will use biochemistry, genetics, molecular biology and mass spectrometry, to idea the factors associated with RNA pol II transcription complexes at different stages of the transcription cycle. I have developed an immobilized template system with which I can rapidly isolate functional transcription complexes from yeast nuclear extracts. After washing the complexes, the associated proteins will be digested with trypsin and analyzed using microcolumn liquid chromatography electrospray ionization mass spectrometry(LC/MS/MS). Proteins will be identified by searching the Saccharomyces cerevisiae genomic sequence database with the Sequest(TM) algorithm. To identify the components that remain at the promoter after initiation of transcription, a similar analysis will be performed on the complexes after addition of nucleotide triphosphates. In addition, covalent modifications of transcription factors, such as phosphorylation and acetylation, will be identified using mass spectral techniques. The timing of the modifications with respect to the transcription cycle will be investigated. Upon identifying novel factors and modifications, the biological relevance of these factors and modifications will be investigated by using molecular generics and biochemistry. Gene knockouts and point mutations will be engineered in S. cerevisiae, to determine the effect of the factor or modification site on cell growth. Nuclear extracts will be prepared from strains carrying conditional mutations in the identified factor to study the role of the factor in in vitro transcription systems. This novel approach to study transcription complexes should yield new insights into the factors involved in transcription and the mechanisms underlying this process. Since regulation of gene expression at the level of transcription is a central process in cancer biology, these studies will make significant contributions to our understanding of this process.